ALS is a rapidly progressive and fatal disease of the motor nervous system. Motor neuron hyperexcitability, manifested as muscle fasciculations and captured in clinical neurophysiological tests1?3, is an early pathological feature that affects both the 10% of familial ALS (fALS) patients who harbor dominant mutations in one of over 25 ALS genes, and the 90% of apparently sporadic ALS (sALS) patients who do not have an affected family member4. However, we do not know whether hyperexcitability causes motor neuron degeneration. Mouse, invertebrate, and human induced pluripotent stem cell (iPSC) models have all shown abnormal motor neuron excitability5?8. However, the mechanistic connection between hyperexcitability and motor neuron degeneration remains unclear6,9,10. In this proposal, we will use iPSCs from fALS patients to derive motor neurons and test the link between hyperexcitability and motor neuron degeneration. We will determine whether multiple fALS mutations increase motor neuron excitability, whether hyperexcitability precedes neurite retraction and cellular stress, and whether hyperexcitability can cause neurite retraction, cellular stress, and motor neuron death. Furthermore, we will examine transcriptomic signatures of multiple fALS lines before, during, and after motor neuron neurite retraction. We anticipate that ion channel transcripts, as identified by gene ontology, will precede neurite retraction in support of an excitotoxicity model. We will then determine whether potential ion channel transcript changes are directly responsible for increased excitability using patch electrophysiology. Finally, using the intersection of RNA-sequencing data sets, we will determine whether RNA-seq signatures from iPSC derived motor neurons overlap with sporadic ALS patient spinal cord tissue. The proposed project is feasible within the three-year time frame because of our experience with high-yield motor neuron differentiation protocols, gene editing, high-content imaging technology, electrophysiology, and computational analyses. The proposed work will determine whether there is a causative link within multiple fALS genetic subgroups between hyperexcitability, and motor neuron degeneration. The project results will have a direct impact on clinical trials attempting to normalize motor neuron excitability in patients. Furthermore, the transcriptomic signatures of multiple fALS lines before, during, and after neurite retraction will allow us to identify common features of motor neuron neurodegeneration that may also apply to sALS, and thus have larger therapeutic implications. Finally, our access to patient tissue allows us to validate specific findings related to excitability and transcriptomic signatures to determine their clinical relevance. We anticipate that this proposal will determine whether hyperexcitability is caused by fALS mutations and contributes to neurodegeneration in ALS, directly impacting ongoing clinical trials and therapeutic development.